Nuclear matrix protein fluid assay

ABSTRACT

Disclosed is a method for evaluating the efficacy of a therapy associated with cell death in a patient undergoing such a therapy. The method involves providing at least two body fluid samples drawn from the patient, wherein a second sample is drawn from the patient after a first sample, and measuring the quantity per unit volume of a body-fluid soluble interior nuclear matrix protein in each of the samples. By comparing the quantity per unit volume of the body fluid-soluble interior nuclear matrix protein in the first sample with the quantity per unit volume of the body fluid-soluble interior nuclear matrix protein in the second sample, it is possible to determine whether there has been an increase or decrease of cell death in the patient during therapy.

This application is a divisional of U.S. Ser. No. 08/112,646, filed Aug.26, 1993, now abandoned, which is a continuation of U.S. Ser. No.07/785,804, filed Oct. 31, 1991, now abandoned.

REFERENCE TO RELATED APPLICATIONS

Related applications include: U.S. Ser. No. 08/456,620, filed Jun. 1,1995, which is a continuation of U.S. Ser. No. 08/112,646, filed Aug.26, 1993, now abandoned, which is a continuation of U.S. Ser. No.07/785,804, filed Oct. 31, 1991, now abandoned; U.S. Ser. No.08/443,630, filed May 18, 1995, a divisional of U.S. Ser. No.08/112,646, filed Aug. 26, 1993, now abandoned, which is a continuationof U.S. Ser. No. 07/785,804, filed Oct. 31, 1991, now abandoned; U.S.Ser. No. 08/195,487, filed Feb. 14, 1994, a continuation of U.S. Ser.No. 07/901,701, filed Jun. 22, 1992, now abandoned; U.S. Ser. No.08/483,924, filed Jun. 7, 1995, which is a continuation of U.S. Ser. No.08/195,487, filed Feb. 14, 1994, a continuation of U.S. Ser. No.07/901,701, filed Jun. 22, 1992, now abandoned; U.S. Ser. No. 08/470,950filed Jun. 6, 1995, a divisional of U.S. Ser. No. 08/195,487, filed Feb.14, 1994, a continuation of U.S. Ser. No. 07/901,701, filed Jun. 22,1992, now abandoned; U.S. Ser. No. 08/466,390 filed Jun. 6, 1995, adivisional of U.S. Ser. No. 08/195,487, filed Feb. 14, 1994, acontinuation of U.S. Ser. No. 07/901,701, filed Jun. 22, 1992, nowabandoned; and U.S. Ser. No. 08/467,781 filed Jun. 6, 1995, a divisionalof U.S. Ser. No. 08/195,487, filed Feb. 14, 1994, a continuation of U.S.Ser. No. 07/901,701, filed Jun. 22, 1992, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates generally to the use of body fluid-solublenuclear matrix proteins as cellular markers. More particularly, theinvention relates to a method of monitoring the degree of cell death ina tissue by monitoring the level of interior nuclear matrix proteinsand/or fragments thereof released from cells in a body fluid-solubleform.

Cell death occurs as a function of healthy tissue homeostasis, as wellas a result of disease or injury to the tissue. Detectable changes inthe rate of cell death in a tissue therefore can provide an indicationof the tissue's status. In normal growing tissue, for example, changesin the rate of cell death can indicate a new developmental stage.Alternatively, a sudden detected change in cell death in adult tissuecan signal an injury to the tissue and also provide information on thetype of injury. Previously, clinical assessment of tissue viability hasrequired presentation of clinical symptoms and a visual inspection ofthe tissue itself, requiring observation of the morphology of cells in atissue sample, and/or an indirect estimation of cell death by measuringchanges in tissue/organ size. While the state of the art ofmorphological observation has advanced significantly, reliance onhistological observation of an abnormality requires that the abnormalcells comprise part of the tissue sample tested. Early stages of amalignancy therefore can be particularly difficult to detect by thismethod. Biochemical assessments of cell or tissue viability alsogenerally have been difficult to interpret, primarily because in manyinstances cell death occurs in individual cells which are intimatelyintermixed with viable cells in a sample.

There exists a need for reliable means of monitoring the degree of celldeath in a tissue which does not rely on histology or tissue biopsy. Aparticularly useful method would be one wherein the rate of cell deathcould be monitored in a body fluid assay. It is anticipated that theability to accurately monitor tissue-specific cell death in a fluidassay would have significant impact on the study of tissue developmentand cell kinetics, as well as on many different clinical applications.For example, the method may be used to monitor the progress of a diseaseor injury associated with cell death, as well as to monitor the efficacyof a therapy for an affected tissue. The method also may be used tomonitor the effect of the therapy on unaffected, normal tissue. Inaddition, the method may be used to evaluate a compound's cytotoxicity.

Recently, methods have been disclosed for extracting and isolating thenormally highly insoluble interior nuclear matrix proteins from cells.Interior nuclear matrix proteins are proteins from within the boundariesof the nuclear matrix, termed "interior" nuclear matrix proteins, whichare substantially free of chromatin proteins, and intermediatefilaments, collectively termed "exterior" nuclear matrix proteins (seePenman et al., U.S. Pat. No. 4,882,268, the disclosure of which ishereby incorporated by reference). In the method, the cell nucleus isisolated, the cytoskeleton proteins and chromatin are removed, the"nuclear matrix" is isolated, and the interior and exterior componentsof the nuclear matrix are separated. The nuclear matrix-intermediatefilament complex comprises a specific fraction of cell proteinconstituting less than five percent of the total protein and six percentof the total DNA of the cell. The interior nuclear matrix comprisesabout one percent or less of the total cellular protein. It containsmany proteins that differ according to cell type, and is highly enrichedwith type-specific antigens including cell-type andtransformation-specific proteins that had not been detected using priorart procedures. It also contains the lamina and nuclear pore complexproteins. The separation method makes use of the unique properties ofthe nuclear matrix to achieve separation from substantially all othercell constituents. The method is simple, rapid, reproducible, achieves ahigh degree of purity, and is applicable to essentially all types ofcells.

The method of Penman et al. has enabled the art to identify and isolatecell type-specific and transformation-specific interior nuclear matrixproteins of sufficient purity to allow antibodies to be made. Theseantibodies then can be used to detect cell type-specific interiornuclear matrix proteins in a sample, as disclosed in U.S. Pat. No.5,273,877, issued Dec. 28, 1993, the disclosure of which is incorporatedherein by reference. As used therein and here, the term "cell-type" isunderstood to refer to cells of various different tissues such asneural, glial, muscle, liver, mesenchymal, and different types ofepithelia and endothelia, as well as malignant cell forms and cellshaving an altered genomic expression profile caused by viral infectionor other factors.

It is an object of this invention to provide a method for monitoring thedegree of cell death in a body fluid assay by monitoring the level ofbody fluid-soluble interior nuclear matrix proteins in a fluid. Anotherobject of the invention is to provide a method for quantifying the bodyfluid-soluble interior nuclear matrix proteins released from cells andpresent in a cell compatible fluid. Still another object of theinvention is to provide a method for evaluating a therapy, or theprogress of a disease associated with cell death by monitoring thedegree of cell death in an affected tissue. Another object of theinvention is to provide a method for assessing the toxicity of compoundsby assessing their ability to induce cell death. Still another object ofthe invention is to provide a method for inducing the release of bodyfluid-soluble nuclear matrix proteins from eucaryotic cells. These andother objects and features of the invention will be apparent from thedescription and claims which follow.

SUMMARY OF THE INVENTION

It now has been discovered that interior nuclear matrix proteins orsoluble fragments thereof are released in soluble form from cellsundergoing cell death. As used herein, "cell death" is understood toinclude both apoptosis ("programmed" cell death), and necrosis.Moreover, it also now has been discovered that these soluble forms ofinterior nuclear matrix proteins can be quantitated in a fluid and usedto monitor the degree, or rate, of cell death in a tissue. The method ofthe invention also may be used to distinguish between types of celldeath occurring. The presence of these proteins and protein fragments insolution in a body fluid soluble form is unexpected as the moleculescomprise part of an insoluble nuclear complex under standardphysiological conditions. The discovery of these proteins released insoluble form from dying cells allows one to use the soluble interiornuclear matrix proteins as biochemical monitors of tissue-specific celldeath in fluid assays. The method is rapid and quantitative and can beused to evaluate the viability of cells and tissue, to evaluate progressof a disease and/or its treatment, to evaluate the cytotoxicity ofunknown compounds and to study the kinetics of cell death. The discoveryalso provides an alternative method for purifying interior nuclearmatrix proteins from cells.

The method of the invention involves detecting the concentration of bodyfluid-soluble interior nuclear matrix proteins or fragments thereofreleased from cells and comparing this concentration to a knownstandard. The concentration of these proteins in a body fluid samplewill be indicative of the degree of cell death in that tissue. Fluidsamples are collected at discrete intervals and detected by means, forexample, of an immunoassay. The concentrations of characteristicproteins then are compared, with changes in concentrations beingindicative of the changes in the rate of cell death. In addition,certain proteins may be identified as cell type-specific and/or as celldeath type-specific. Exemplary body fluids include blood, serum, plasma,urine, semen, spinal fluid, saliva, ascitic fluid, peritoneal fluid,sputum, tissue swabs, and body exudates such as breast exudate.

In one embodiment of the invention, the method may be used to evaluatethe progress of a disease. For example, the method may be used tomonitor the progress of a malignancy such as, for example, a carcinoma,adenoma, sarcoma, lymphoma, or myeloma. Here the rate of cell death in amalignant tissue may be monitored by quantitating the level of solublenuclear matrix protein released from the malignant cells. Alternatively,the method also may be used to monitor the progress of tissue disordersresulting in altered cell death, such as results from tissue atrophy,hyperplasia, cirrhosis, hypoxia, ischaemia and benign tumor growths.Injured tissue also can be assessed by this method, including directcell trauma such as from membrane-active chemicals and toxins orresulting from direct physical trauma, such as, for example,hyperthermia, hypoxia and ischemia/reperfusion, radiation orcomplement-mediated autolysis.

In another embodiment of the invention, the method may be used tomonitor the efficacy of a therapy. Here a therapeutic agent or procedure(e.g., radiation therapy) is administered to a patient and, thereafter,the concentration of soluble nuclear matrix proteins or fragments aredetected in body fluid samples drawn from the patient at predeterminedintervals. These concentrations then are compared to each other and tothose in samples tested before administration of the therapy. Thechanges in concentration of one or more interior nuclear matrix proteinsdetected among the samples compared will be indicative of the therapy'sefficacy. For example, a therapeutic agent capable of selectivelydestroying malignant cells can cause an increase in soluble nuclearmatrix proteins released from the tumor cells, followed by a reductionin the level of these proteins detected as the number of malignant cellsfall. The method also may be used to monitor the effect of a giventherapy on the viability of normal, unaffected tissue. For example, anumber of cancer therapies, including radiation and chemotherapy, targetrapidly proliferating cell populations and therefore can affect normallyproliferative cell populations such as bone marrow progenitor cellpopulations and intestinal epithelial cells. The effect of the therapyon these cell populations can be evaluated by monitoring the level ofsoluble interior nuclear matrix protein released from these cells. It isanticipated that a single body fluid sample can provide information onthe viability of both the affected tissue and normal tissue providedthat cell-type specific proteins can be monitored for each tissue.Similarly, the method also may be used to evaluate the efficacy ofcomplementary therapeutic agents designed to protect normal tissue fromthe effects of the therapy.

In yet another embodiment of the invention the method may be used toevaluate the type of cell death occurring and to study its kinetics. Forexample, nuclear matrix proteins or protein fragments may be identifiedthat are preferentially released in soluble form by cells undergoingeither apoptosis or necrosis. The rate of release of these proteins orprotein fragments then may be monitored to investigate cell deathkinetics.

In still another embodiment of the invention the method may be used tomonitor the status of a cell culture and/or to assess the cytotoxicityof a compound by monitoring the levels of soluble interior nuclearmatrix proteins or protein fragments released from these cells. Finally,the method also may be used to induce release of body fluid-solubleinterior nuclear matrix proteins from intact cells, using compoundscapable of inducing apoptosis, such as, for example, cytokines. Aparticularly useful cytokine is TNF (Tumor Necrosis Factor). Thisembodiment of the invention may be used to enhance identification ofparticular interior nuclear matrix proteins. It further may be used aspart of a protocol to isolate soluble interior nuclear matrix proteinsor protein fragments. Novel nuclear matrix proteins may be obtained bythis method, as well as known or novel proteins useful as antigenicdeterminants in antibody production. Finally, the method of theinvention may be useful as part of a protocol to identify candidatecompounds useful as cancer chemotheapeutic agents (see infra.)

These and other embodiments and features of the invention will beapparent from the specification, drawings and claims, which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (1.1 and 1.2) lists the levels of body fluid-soluble interiornuclear matrix protein quantitated in various normal and malignanttissue sample supernatants;

FIG. 2 lists the levels of body fluid-soluble interior nuclear matrixproteins quantitated in sera isolated from cancer patients and normalblood donors;

FIG. 3 is a graphic representation of the soluble nuclear matrixproteins detected by the 22/18 and 41/7 antibody combinations followingthe release of these nuclear matrix proteins from necrotic and apoptoticcells;

FIG. 4 (A and B) are graphic representations of the effect of TNF (tumornecrosis factor) on soluble nuclear matrix protein released fromcultured cells;

FIG. 5 is a graph correlating nuclear matrix protein release with celldensity;

FIG. 6 is a graph correlating TNF dose with the release of solublenuclear matrix protein detected by the 41/7 antibody combination;

FIG. 7 is a graphic representation of the effect of Doxorubicin onsoluble nuclear matrix protein release and cell viability.

DETAILED DESCRIPTION

In the description which follows, the generalized method for monitoringcell death by quantitating the body fluid-soluble interior nuclearmatrix proteins released from cells into a fluid is disclosed. Themethod involves the selection of interior nuclear matrix proteins toform a substantially pure preparation of interior nuclear matrixproteins, herein referred to as a NM preparation, and the use of thispreparation to generate nuclear matrix protein-specific antibodies.Preferably the antibodies are cell-type specific. The antibodies alsomay be cell death-type specific. Next, methods are disclosed fordesigning assays useful for monitoring cell death with these antibodiesin different fluid assays, including serum. Methods also are disclosedfor inducing the release of these proteins in soluble form from cells.

Biologically relevant mechanisms of cell death generally are classifiedas either apoptosis or necrosis, primarily on the basis of histologicalstudies. The two mechanisms generally are distinguished functionally andmorphologically. Apoptosis generally is understood to characterize thecoagulative necrosis of single cells, so-called "programmed" cell death.It characteristically affects individual cells in an asynchronousfashion, and generally is not accompanied by an inflammatory reaction.Apoptosis is involved in the programmed focal elimination of cells thataccompanies embryonic and fetal development, and in regulating the sizeof organs and tissue in adult life. Apoptosis also has been identifiedin pathological tissue atrophy, as well as in a variety of malignantneoplasms, and following exposure to a range of injurious agents, anumber of which are associated with DNA damage, e.g., radiation,hyperthermia, and a variety of carcinogens and cancer-chemotherapeuticagents. Cell-mediated immune reactions also are thought to induceapoptosis, and this mechanism of cell death also is associated withdiseases highlighted by cell-mediated immune destruction of tissues,including so-called auto-immune diseases, cirrhosis, and a number ofviral diseases including hepatitis and the like.

By contrast, necrosis generally is understood to refer to theprogressive degeneration of cell structure that occurs after deathcaused by severe, injurious changes in environmental conditions.Necrosis typically affects groups of contiguous cells and aninflammatory reaction usually develops in the adjacent viable tissue.Among the many known examples of necrosis are cell destruction followingsevere hypoxia and ischemia, in tissue undergoing autolysis in vitro,and cell destruction following severe hyperthermia, or exposure tonumerous membrane-active chemicals, toxins and reactive oxygenmetabolites.

Morphologically, necrotic cells tend to appear swollen, witheosinophilic cytoplasm and indistinct cell boundaries. Cells undergoingapoptosis are characterized by chromatin condensation and by theformation of membrane-bound apoptotic bodies.

All eukaryotic cells, both plant and animal, have a nucleus surroundedby the cell cytoplasm. The nucleus contains DNA complexed with proteinand termed chromatin. The chromatin, with its associated proteins,constitutes the major portion of the nuclear mass. The chromatin isorganized by the internal protein skeleton of the nucleus referred toherein as the interior nuclear matrix. A method for extracting andselectively purifying the highly insoluble interior nuclear matrixprotein from cells recently has been discovered (see U.S. Pat. No.4,882,268 incorporated herein by reference). It also has been discoveredthat certain interior nuclear matrix proteins are tissue and celltype-specific and that their expression also can be altered in atransformation- or disease-specific manner in affected cells. Thisalteration in expression pattern makes these proteins ideal markers fordetecting and identifying abnormal cell growth, particularly fordetecting malignancies, (see U.S. Pat. No. 5,273,877 issued Dec. 28,1993, incorporated herein by reference). Following the methods disclosedin the U.S. Pat. Nos. 4,882,268 and 4,885,236 patents, interior nuclearmatrix proteins can be selectively purified and used as antigenicdeterminants to raise antibodies which may be used to detect thepresence of these proteins in cell samples or as part of the cellulardebris from destroyed cells.

A detailed description for isolating interior nuclear matrix proteins isdisclosed in U.S. Ser. No. 214,022 filed Sep. 30, 1988 and incorporatedby reference herein (see supra). Broadly, the isolation procedureinvolves the following:

1. Isolation and separation of cells.

2. Separation of soluble cell proteins from the nucleus and cytoskeletonby extraction of membrane lipids and soluble proteins with a non-ionicdetergent-physiological salt solution.

3. Separation of cytoskeleton proteins from the nucleus bysolubilization of the insoluble cell material from step 2 in either0.25M ammonium sulfate, pH 6.8, a detergent-sodium deoxycholatesolution, or other gentle extraction buffer.

4. Separation of chromatin from the nuclear matrix by digestion of theinsoluble material from step 3 with DNAse I and RNAse in a physiologicalbuffer, and elution of the DNA-containing nucleosomes with 0.25Mammonium sulfate solution buffered to pH 6.8 or other gentle extractionbuffer.

5. Separation of the interior nuclear and "exterior" nuclear matrixproteins by dissolution of the insoluble material from step 4 in abuffer containing between 5 and 10M urea, preferably 8M urea, or othersuitable solubilizing agent, and aggregation of the exterior proteins bydialysis into physiological buffer.

In a variation of this procedure, and as used herein, the cytoskeletonproteins and chromatin are removed together by digesting the insolublematerial from step 2 with DNAse and RNAse, then extracting with 0.25Mammonium sulfate at ph 6.8. Further purification of the interior nuclearproteins can be performed using standard methods known to those skilledin the art, including HPLC, FPLC, chromatofocusing and the like.

The matrix preparation is biochemically and morphologically pure byseveral biochemical criteria. Freedom from contamination permits a clearand detailed analysis of the matrix proteins by gel electrophoresis. Itpreviously has been shown that electropherograms of a NM preparationshow markedly different patterns in different cell types. These patternsare unique, specific and reproducible. It further has been discoveredthat this pattern allows selection of one or more proteinscharacteristic of the cell type and useful as antigenic determinants.(See, for example, U.S. Pat. No. 4,882,268 and U.S. Pat No. 5,273,877)As shown therein, individual matrix proteins useful as cell-typediscriminators often comprise less than 0.01% of the cell's totalprotein. Antibodies raised against these proteins then can be used toidentify cell-type specific nuclear matrix proteins in samples.Moreover, antibodies raised against these proteins also may be used todistinguish the type of cell death occurring (see infra.)

As indicated above, the method of the invention involves monitoring celldeath by quantitating the soluble interior nuclear matrix proteinsreleased from cells into a fluid. As will be appreciated by thoseskilled in the art, any means for specifically identifying andquantifying the protein is contemplated. The current state of the artfor identifying proteins in solution is by means of an immunoassay,wherein an antibody capable of binding specifically to the protein ofinterest is used to identify the protein in solution and the amount ofbound complex formed then is determined.

Antibody methodologies are well understood and described in theliterature. A more detailed description of their preparation can befound, for example, in Practical Immunology, Butt, W. R., ed., MarcelDekker, New York, 1984. Broadly, antibodies may be raised against one ormore proteins in a NM preparation by immunizing a suitable animal withan immunogenic preparation under conditions sufficient to induceantibody production in that animal. Monoclonal antibodies then can beobtained by fusing suitable antibody producing cells such as spleen orlymph node cells to myeloma cells and screening the fusion products fornuclear reactivity against the immunogen source (e.g., cell line orparticular cell type determinant) using standard techniques. A detailedprotocol for screening for particular cell type determinants can befound in U.S. Ser. No. 214,022, incorporated by reference hereinabove.

The currently preferred method for quantitating body fluid-solublenuclear matrix proteins is by detecting the proteins with interiornuclear matrix protein-specific antibodies. The antibodies may bemonoclonal or polyclonal in origin, and may be produced by standardmethodologies. The nuclear matrix proteins used as immunogens may beprepared by the method disclosed by Penman et al. (U.S. Pat. No.4,882,268, see infra). Alternatively, interior nuclear matrix proteinsmay be isolated by inducing their release in soluble form from cells asdescribed herein, and selectively extricating them from the supernatant.Proteins or protein fragments also may be recombinantly expressed fromnucleic acids using standard recombinant DNA technology. Antibodies toone or more of these proteins then are raised using standard methods.The antibodies then are exposed to the body fluid sample underconditions sufficient to allow specific binding of the antibody to itsspecific nuclear matrix protein, and the amount of complex formed thendetected.

A variety of different forms of immunoassays currently exist, all ofwhich may be adapted to quantitate body fluid-soluble interior nuclearmatrix proteins released from cells. Of the various immunoassay formatsavailable, one of the most sensitive is the sandwich technique. In thismethod, two antibodies capable of binding the analyte of interest areused: one immobilized onto a solid support, and one free in solution,but labeled with some easily detectable chemical compound. Examples ofchemical labels that may be used for the second antibody includeradioisotopes, fluorescent compounds, and enzymes or other moleculeswhich generate colored or electrochemically active products when exposedto a reactant or enzyme substrate. When samples containing analyte(e.g., body fluid-soluble interior nuclear matrix protein or proteinfragments) are placed in this system, the analyte binds to both theimmobilized antibody and the labelled antibody. The result is a"sandwich" immune complex on the support's surface. The analyte isdetected by washing away nonbound sample components and excess labeledantibody and measuring the amount of labeled antibody complexed toanalyte on the support's surface. The sandwich immunoassay is highlyspecific and very sensitive, provided that labels with good limits ofdetection are used. A detailed review of immunological assay design,theory and protocols can be found in numerous texts in the art,including Practical Immunology, Butt, W. R., ed., Marcel Dekker, NewYork, 1984.

Immunoassay design considerations include preparation of antibodies(monoclonal or polyclonal) having sufficiently high binding specificityfor their antigen that the specifically-bound antibody-antigen complexcan be distinguished reliably from nonspecific interactions. The higherthe antibody binding specificity, the lower the antigen concentrationthat can be detected. The choice of tagging label for the secondantibody also will depend on the detection limitations desired. Enzymeassays (ELISAs) typically allow detection of a colored product formed byinteraction of the enzyme-tagged complex with an enzyme substrate.Alternative labels include radioactive or fluorescent labels. The mostsensitive label known to date is a chemiluminescent tag whereinteraction with a reactant results in the production of light. Usefullabels include chemiluminescent molecules such as acridium esters orchemiluminescent enzymes where the reactant is an enzyme substrate.When, for example, acridium esters are reacted with an alkaline peroxidesolution, an intense flash of light is emitted, allowing the limit ofdetection to be increased 100 to 10,000 times over those provided byother labels. In addition, the reaction is rapid. A detailed review ofchemiluminescence and immunoassays can be found in Weeks, et al., (1983)Methods in Enzymology 133:366-387. Other considerations include the useof microtiter wells or column immunoassays. Column assays may beparticularly advantageous where rapidly reacting labels, such aschemiluminescent labels, are used. The tagged complex can be eluted to apost-column detector which also contains the reactant or enzymesubstrate, allowing the subsequent product formed to be detectedimmediately.

The invention may be better understood from the following nonlimitingexamples wherein interior nuclear matrix proteins are prepared from twodifferent cell lines (breast tumor and cervical tumor cells), polyclonaland monoclonal antibodies are made and tested for reactivity andsensitivity, and immunoassays are performed on sera, and tissue and cellculture supernatants. Apoptosis generally is induced herein by placingcells in serum-free media, by exposure to a cytokine (e.g., TNF) or byexposing cells to particular cytotoxic agents. Necrosis is inducedherein by freeze thawing (hyperthermia.) Using the methods disclosedherein, and variations of these methods, cell death can be quantitatedand monitored in vivo and in vitro by analyzing body fluids or bodyfluid compatible solutions, e.g., extracellular culture media, forsoluble nuclear matrix proteins or protein fragments. Generally, themethod involves preparation of a standard ("dose response") curve forthe nuclear matrix protein to be monitored, and against which samplescan be prepared. As indicated earlier, the status of multiple differenttissues also may be assessed in a single fluid sample, by monitoring theconcentrations of different cell-type specific nuclear matrixdeterminants in the sample.

Also disclosed is a method for inducing release of interior nuclearmatrix proteins in soluble form from cells, using compounds capable ofinducing cell death. Currently preferred compounds include cytokinessuch as TNF which induce apoptosis. Inducing release of interior nuclearmatrix proteins from cells into a fluid can enhance evaluation of atissue's status. It also is envisioned that the method may be useful aspart of a protocol for identifying and purifying novel interior nuclearmatrix proteins, for preparing proteins and protein fragments for use asantigens in antibody preparations, and for identifying candidatecompounds useful in cancer chemotherapy (see infra.)

I. Antibody Development

A. Isolation of Nuclear Matrix

Nuclear matrix proteins are isolated herein essentially according to themethod of Penman and Fey, disclosed in U.S. Pat. No. 4,882,268, thedisclosure of which is incorporated herein by reference. Briefly,desired cell lines are grown to confluency in Dulbecco's ModifiedEagle's medium (DME) (Mediatech, Washington, D.C.) with 10% fetal calfserum (Hazelton) in T225 flasks. The media then is removed from theflask and the cells washed twice with phosphate buffered saline (PBS)(0.1M Phosphate, 0.15M NaCl pH 7.4). The washed cells then are removedfrom the flask by incubation with 0.1% tryspin/EDTA (Irvine Scientific,Santa Ana, Calif.) in PBS at 37° C. for 15 minutes. The resulting cellsuspension is poured into 10 ml of the original media and pelleted bycentrifugation at 1500×g for 15 min. The media then is aspirated off andthe cells resuspended, and counted. The cells are again pelleted andextracted twice with cold RSB/Majik buffer (0.1M NaCl, 10 mM MgCl₂, 10mM TRIS, 0.5% Na Deoxycholate, 1.0% Tween-20, 1.2 mM PMSF pH 7.4) andspun at 2100×g for 15 min at 4° C. 10 ml of RSB/Majik is used for every1×10E7 cells. At room temperature the extracted cells are resuspended indigestion buffer (50 mM NaCL, 300 mM sucrose, 0.5% Triton X-100, 10 mMPIPES, 3 mM MgCl₂, 1 mM EGTA, 1.2 mM PMSF). DNase I and RNase A(Boehringer Mannheim, Germany) are added to a concentration of 100 ug/mland the solution incubated with agitation for 20-40 min. Sufficient 2Mammonium sulphate is added to make a final concentration of 0.25M andafter 5 min the solution is centrifuged at 3700×g for 15 min. Theresulting pellet is resuspended in disassembly buffer (8M urea, 20 mMMES, 1 mM EGTA, 1.2 mM PMSF, 0.1 mM MgCl₂, 1% 2-mercaptoethanol pH 6.6)and dialyzed against 4 liters of assembly buffer (0.15M KCl, 25 mMImidazone, 5 mM MgCl₂, 2 mM dithiothreitol, 0.125 mM EGTA, 0.2 mM PMSFpH 7.1) overnight at room temperature. The resulting suspension is thencentrifuged in an ultracentrifuge at 100,00 g for one hour. Thesupernatant containing the interior nuclear matrix proteins (referred toherein as the NM preparation) then can be stored frozen at -80° C. untilrequired.

For the examples disclosed herein, interior nuclear matrix proteins wereisolated from three human tumor cell lines, all available through theAmerican Type Culture Collection (ATCC, Rockville, Md.): T-47D (a breasttumor cell line); and ME-180 and CaSki (both cervical tumor cell lines).

B. Immunization Protocols

1. Polyclonal Antibodies

Polyclonal antisera can be raised against the NM preparation usingstandard methodologies, such as those disclosed in numerous textsavailable in the art and known to those generally skilled in the art. Inthis example, rabbits were immunized with the NM preparations first inFreund's complete adjuvant (Gibco, Grand Island, N.Y.) and then everymonth with NM with incomplete adjuvant for three months. Rabbit sera andsera from mice prior to fusion were used as polyclonal antisera and wereshown by standard western blot technique to be reactive with the NMpreparations.

2. Monoclonal Antibodies

Monoclonal antibodies can be raised against the NM proteins also usingstandard methodologies, such as those disclosed in numerous textsavailable in the art and known to those generally skilled in the art. Inthis example, Balb/c by J (The Jackson Laboratory, Bar Harbor, Me.) micewere immunized with the nuclear matrix protein preparations in thefollowing manner:

(a) Lymph Node Protocol: Animals are injected in the two hind foot padsand hind quarters using the following protocol: day 1, Antigen andcomplete Freund's Adjuvant; day 4, antigen and saline in hindquartersonly; day 13, the animal is sacrificed and the popliteal lymph nodesremoved for fusion.

(b) Spleen Protocol: Animals are injected every two weeksintraperitoneally for 6-8 injections, then boosted intravenously oncefour days prior to sacrifice and removal of the spleen. Injection #1 iswith complete Freund's adjuvant, #2 is incomplete adjuvant, andsubsequent injections are with saline.

C. Fusions

Hybridomas of the spleen or lymph node cells are fused essentiallyaccordingly to the method of Kohler and Milstein (1975) Nature 256:495,the disclosure of which is herein incorporated by reference. Briefly, asuspension of spleen or lymph node cells are fused with cells of a mousemyeloma cell line using polyethylene glycol (PEG, Boehringer Mannheim).Resulting positive hybridomas then are cloned a minimum of three timesto achieve monoclonicity. Mouse myeloma cell lines used herein wereSP2/0-Ag14 or P3X63Ag8.653 (ATCC, Rockville, Md.)

1. Antibody Screening (Cell Assay/Reactivity Assay)

Growth positive wells from the hybridomas can be screened for nuclearreactivity against the cell line that is the source of the immunogenusing the minifold I dot blot apparatus (Schleicher & Schuell, Keene,N.H.) and standard procedures. Briefly, a 4.5" by 3" piece ofnitrocellulose 0.45 μm filter membrane (Bio-Rad, Richmond, Calif.) andtwo pieces of similar size Whatman 3 MM paper are soaked in distilledwater and PBS for 10 min each and then assembled in the dot blotapparatus. 1×10E6 cells stored frozen in 10% DMSO culture media arewashed in PBS and 100 μl per well of a 10 ml cell suspension are addedwith the vacuum suction on. The cells then are washed with 100 μl ofdigestion buffer, 400 μl of PBS with 0.05% Tween-20, 100 μl of 0.5%nonfat dried milk (Carnation) in PBS (to block nonspecific binding), and400 μl of PBS Tween-20. With the vacuum turned off, 100 μl ofsupernatant from growing hybrids are incubated with the cells for 30min. The supernatant then is removed via vacuum suction and the cellsagain washed with 400 μl of PBS Tween-20. With the vacuum off, 100 μl ofa goat anti-mouse biotin conjugate in 1% horse serum PBS (e.g.,VECTASTAIN, Vector labs, Burlingame, Calif.) is added to each well andincubated for 15 min. The cells then are washed with 400 μl of PBSTween-20 and incubated with the Vectastain Avidin-HRP substrate for 3min. The reaction is stopped with tap water and the membrane removedfrom the apparatus and soaked in water. The membrane then is sandwichedbetween two glass plates with microscope slide resin and examined underthe microscope. Wells showing positive nuclear staining are consideredpositive and these hybrids are chosen to be further cloned.

2. Tissue Immunohistochemistry

Positive clones can be evaluated for human tissue reactivity usingcryostat cut frozen tissue sections and the avidin/biotin peroxidestaining procedure. (Vectastain Elite ABC kit, Vector Laboratories,Burlingame, Calif.)

3. Ascites Production

Mice are pristane (2,6,10,14 Tetramethylpentadecane) primed by injecting0.5 ml pristane into the peritoneal cavity. Seven to ten days later5×10E6 cells in one ml of serum free media are injected into theperitoneal cavity. Ascites fluid then is collected from each mouse twiceand pooled. Monoclonal anti-NM antibodies are purified from mouseascites by protein G purification (Genex Corp., Gaithersburg, Md.) andstored at 4° C. until required.

D. Biotinylation of Antibodies

Antibodies can be labelled following standard techniques. In theseexamples, antibodies are biotinylated conventional procedures. Briefly,purified antibodies (herein, monoclonal anti-NM antibodies purified frommouse ascites) are dialyzed overnight against 0.1M sodium bicarbonate pH8.4 and the concentration adjusted to 1.0 mg/ml. Biotin-X-NHS (0.5 mlCalBiochem, Inc., San Diego, Calif.) 1.5 mg/ml in DMSO, then is added to5 ml of the antibody solution and reacted for 2 hr at room temperaturewith rocking in the dark. 2M Tris-HCL pH 8.0 (0.5 ml) is added andincubated for a further 30 min. PBS-merthiolate (0.01%) containing 1%BSA then is added (5 ml) and the mixture dialyzed three times againstthe PBS-Merthiolate.

II. Assays

A. Sandwich Immunoassay (ELISA)

A standard immunoassay can be performed to generate dose response curvesfor antigen binding, for cross reactivity assays, and for monitoringassays. The data is generated with a standard preparation of NM antigen,and is used as the reference standard when body fluids are assayed. Inthese examples both ELISAs and radioummunoassays were performed.

1. Immunoassay (Well Assay)

Microtitre plates (Immulon II, Dynatech, Chantilly, Va.) are coated withpurified antibody at 5 to 15 ug/ml in PBS at pH 7.4 for 1 hr orovernight and then washed 3× with 300 μl PBS. The plates then areblocked with 10% normal goat serum in PBS for 1 hr at room temp andwashed 3× with 300 μl of PBS.

Here, samples were assayed by pipetting 100 μl of sample per well, andincubating for 1 hr at RT. The wells were washed with 3×300 μl PBS. 100μl of 1.25 to 10 μg/ml of a biotinylated antibody was added to eachwell, incubated for 1 hr at RT and washed with 3×300 μl of PBS. 100 μlof a 1:1000 dilution of streptavidin-horseradish peroxidase conjugate(The Binding Site Ltd., Birmingham, UK) was added to each well andincubated for 1 hr and then washed with PBS. 100 μl of peroxidasesubstrate (citrate, phosphate, OPD-H₂ O₂) was added to each well andincubated for 20 min. The reaction was stopped by adding 50 μl of 1M H₂SO₄ to the wells. The optical density was read on a plate reader at 490nm.

Concentrations of NM antigen were determined by preparing a referenceconcentration of NM and preparing a standard dilution curve to comparewith the unknown samples.

2. IRMA (Immunoradiometric Assay)

(a) Iodination of Streptavidin

10 μg of streptavidin (Sigma, Inc., Cincinnati) in 2 μl of 0.05Mphosphate pH 7.4 was added to 10 μl of 0.25M phosphate pH 7.4 in amicrocentrifuge tube and 1 mCi of ¹²⁵ I (NEN-DUPONT, Wilmington, Del.)in 10 μl was added. Immediately 10 μl of 100 mg chloramine-T trihydrate(Sigma, Inc.) in 50 ml of distilled water was added, mixed, and reactedfor 25 sec. The reaction was stopped by mixing for 20 sec with 50 μl of40 mg Cysteamine (2-mercaptoethlyamine) (Sigma, Inc.) and 5 mg KI in 50ml of 0.05M phosphate pH 7.4. 0.5 ml of 1% BSA in PBS pH 7.4 was addedand the material was fractionated on a 10 ml sephadex G-100 column(Pharmacia, Sweden) pre-equilibrated with the BSA PBS buffer. 30 by 0.5ml fractions were collected and 10 μl was diluted to 1 ml of the BSA/PBSbuffer for each fraction. 100 μl of the diluted fraction was counted ona LKB gamma counter set for ¹²⁵ I. The specific activity was calculatedand routinely fell between 85 to 100 uCi/ug. The mid fractions of theprotein peak were used in the sandwich immunoassay.

(b) Sandwich Radioimmunoassay

The microtitre breakaway wells (Immulon II Removawell strips, Dynatech,Chantilly, Va.) are coated and blocked as in the ELISA assay. Thesamples, standard or sera, are routinely measured by incubating 100 μlin the wells for 1 hr at RT washing on a plate washer with 3×300 μl ofPBS and then incubated with the biotinylated antibody (2-10 μg/ml in 10%goat serum) for 1 hr at RT and washed again. The bound biotinylatedantibody is detected with the ¹²⁵ I-streptavidin. 200,000 to 300,000 cpm(77% counter efficiency) in 100 μl is added to each well and incubatedfor 1 hr at RT and washed again. The bound fraction is detected bycounting the radioactivity in an LKB gamma counter. The concentrationcan be determined by comparing the counts obtained against a referencepreparation.

B. Dot Blot Detection of NM

Antibody reactivity with NM proteins can be assessed by dot blotdetection assays, using standard methodologies and apparatus (e.g.,Schleicher & Schuell). Nitrocellulose membranes are soaked in Trisbuffered saline, (TBS, 50 mM TRIS, 150 mM NaCl, pH 7.6) and NMpreparation applied at varying concentrations of protein to a series ofwells and incubated for 1 hr at room temperature (e.g., T-47D NMsupernatant at 10 μg/ml, 1 μg/ml and 100 ng/ml). The blocked wells thenare washed with 2×200 μl of TBS and then blocked with 100 μl 10% normalgoat serum in TBS for 1 hr at room temperature. The blocked wells thenare washed again with 2×200 μl of TBS and 100 μl of culture supernatantcontaining nuclear reactive antibody to be tested is added to theirrespective wells and incubated for 1 hr at room temperature. The wellsthen are washed with 2×200 μl of TBS and 100 μl of a dilution series ofalkaline phosphatase conjugated goat anti-mouse IgG (Bio-Rad, Richmond,Calif.) (e.g., 1:1000, 1:5000, or 1:10000) added to the relevant wellsand incubated for 1 hr. The wells then are washed with 2×200 μl of TBSfollowed by addition of enzyme substrate (BCIP/NBT, Kirkgaard and Perry,Gaithersburg, Md., e.g., 100 μl) in Tris buffer containing Levamisole(Vector, Inc., Corpus Christi, Tex.) A fifteen minute incubationgenerally is sufficient. The reaction can be stopped by washing withdistilled water and the product detected.

III. Soluble Antigen Preparation from Dying Cells (Serum Deprivation)

Cell lines are grown to confluency in tissue culture flasks by standardculturing techniques. The media then is replaced with serum-free mediaand the cells placed in a 37° C. incubator with 5% CO₂ for 7 to 14 days,depending on the cell line. At the end of the incubation the media iscollected and centrifuged at 14,000×g to remove cellular debris, and thesupernatant stored frozen.

Where human tissue is used, normal and tumor, the soluble interiornuclear matrix proteins can be released in a similar fashion. Tissue isremoved from a donor, flash frozen in liquid nitrogen within 10 min to 4hrs after removal and stored at -70° C. until required. When ready to beused, the tissue is chopped into 0.1 to 0.3 cm cubes as it thaws usingaseptic techniques in a laminar flow hood and placed in a T150 flaskcontaining serum free media containing Fungizone and gentamycin. Ingeneral, 2-4 g of tissue are used per 100 ml media in the T150 flask.The flask containing the tissue then is incubated for 4-7 days at 37° C.with 5% CO₂. After incubation the media is collected from the flasks,centrifuged at 14,000×g for 20 min and the supernatant stored at -20° C.until needed.

IV. Representative Assays

Reactivity Assays

Immunoblot studies for anti-nuclear matrix reactivity were performedwith monoclonal antibodies raised against T-47D nuclear matrix toidentify those having strong reactivity with the T-47D antigen. Amongthose identified were three antibodies referred to herein as 200-4,203-37, and 304-41. All antibodies exhibited a positive reaction. Theintensity of reaction was best at the higher concentrations of secondaryantibody, and no reaction was observed in the absence of antigen.

Using the same assay conditions these three antibodies, 200-4, 203-37,and 304-41, all showed reactivity with the supernatant from T-47D dyingcells, but not with the media alone, demonstrating the release ofsoluble antigen into the cell culture supernatant. Anti-KLH (keyholelimpet hemocyanin) monoclonal antibody used as a negative control,showed no reactivity with the spent supernatant confirming thespecificity of the antibody reactions. Similar results were obtainedwith polyclonal rabbit sera raised against the nuclear matrix proteins.

Table I below displays the binding results for assays performed withdifferent antibodies raised against the two different cervical tumorcell line NM antigen preparations (ME-180 and CaSKi). The 100-seriesantibodies are those raised against the ME-180 NM immunogen; the300-series are those raised against CaSKi-NM immunogen.

                  TABLE I                                                         ______________________________________                                               SOLUTION Ab                                                            CAPTURE Ab                                                                             107-7   302-18  302-22                                                                              302-29                                                                              302-47                                                                              307-33                             ______________________________________                                        107-7    NO      NO      NO    RX    NO    RXN                                         RXN     RXN     RXN         RXN                                      302-18   NO      RXN     RXN   RXN   RXN   RXN                                         RXN                                                                  302-22   NO      RXN     NO    NO    NO    RXN                                         RXN             RXN   RXN   RXN                                      302-29   NO      RXN     NO    NO    NO    RXN                                         RXN             RXN   RXN   RXN                                      302-47   NO      NO      NO    NO    NO    NO                                          RXN     RXN     RXN   RXN   RXN   RXN                                307-33   NO      NO      NO    RXN   NO    NO                                          RXN     RXN     RXN         RXN   RXN                                ______________________________________                                    

As can be seen from the table, twelve of the thirty-six combinationstested result in a positive reaction. A positive reaction means that thetwo antibodies react with different epitopes on the same molecule. Onlyone antibody, 302-18, reacted in combination with itself.

Dose Response Assays

The first sandwich assay was obtained using antibodies 200.34 and 200-4on nuclear matrix proteins isolated by the method of Penman and Fey(Table II) and on cell culture supernatant from dying cells (Table III).The cell line T-47D was used as the source of antigen for bothexperiments and demonstrated that a dose response curve can be obtainedwith these assay conditions.

Table II shows the data generated using a standard ELISA immunoassay andpurified NM, isolated by the method of Penman et al. Table III shows thedata generated under the same conditions, but using the supernatant ofdying cells as the antigen source. The cell line T-47D was used as theantigen source for both experiments and two antibodies, previously shownto have strong reactivity with the T-47D antigen by dot blot assay, wereused (Ab 200-34, solid phase; Ab 200-4 as soluble antibody).

                  TABLE II                                                        ______________________________________                                        Protein Concn.                                                                            OD                                                                in NM prep. rep 1        rep 2  mean                                          ______________________________________                                        10     μg/ml 0.186        0.187                                            1      μg/ml 0.036        0.032                                                                              0.034                                       0.1    μg/ml 0.021        0.009                                                                              0.015                                       0.0             0.00         0.003                                                                              0.001                                       ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Concentration                                                                 of supernatant   Mean OD  SD                                                  ______________________________________                                        Undiluted        0.150    0.015                                               1:2              0.071    0.010                                               1:4              0.026    0.003                                               1:8              0.013    0.005                                               No Sup                                                                        2:1              0.401    0.015                                               undiluted        0.145    0.006                                               1:2              0.05     0.002                                               1:4              0.017    0.003                                               1:8              0.003    0.002                                               No Sup           0.000                                                        ______________________________________                                    

The data show that reliable dose response curves can be generated usingthese assay conditions to quantitate soluble NM antigen in solution.Following this protocol, other antibody combinations can be tested fortheir ability to detect and quantitate body fluid-soluble nuclear matrixproteins and protein fragments.

Dose response evaluation results of two of these antibody combinationsare shown in Table IV, below, using ME-180 cell culture supernatant asthe antigen source. Each assay shows dose dependent detection of antigenin the tissue culture supernatant, demonstrating the ability of theassay to quantitate soluble interior nuclear matrix protein releasedfrom dying cells.

                  TABLE IV                                                        ______________________________________                                        Concentration                                                                 of supernatant   Mean OD  SD                                                  ______________________________________                                        Antibody 107-7 solid phase,                                                   302-29 soluble antibody, ME-180 supernatant                                   2:1              0.501    0.013                                               undiluted        0.274    0.018                                               1:2              0.127    0.006                                               1:4              0.067    0.006                                               1:8              0.035    0.009                                                1:16            0.021    0.007                                               No Sup           0.000                                                        Antibody 107-7 solid phase,                                                   307-33 soluble antibody, ME-180 supernatant                                   3:1              0.906    0.009                                               3:2              0.456    0.011                                               3:4              0.216    0.007                                               3:8              0.099    0.005                                                3:16            0.052    0.002                                                3:32            0.031    0.005                                               No Sup                                                                        ______________________________________                                    

Next, interior nuclear matrix protein quantification was tested insupernatant from a variety of dying tumor tissues. Here, tumor andnormal tissues were allowed to die in media as described supra.Supernatants were assayed in various configurations of sandwich assays.The results are shown in FIG. 1, where all values are in units/gm, usingME-180 antigen as standard. As can be seen from FIG. 1, antigen isreleased from each of the dying tissues, and the three assays aremeasuring different antigens. As expected, the increased cell death intumor tissue is reflected in a higher average antigen value quantitatedin cancer tissue versus normal tissue. In addition, significantdifferences in antigen quantities are seen in the different tissuesources, indicating that the soluble antigen quantities present in thesupernatant vary in a cell-type specific manner.

FIG. 2 shows the results of an analogous experiment performed usingserum samples from cancer patients and normal blood donors. As for FIG.1, ME-180 cell antigen was the standard. Results are reported inunits/ml. A control experiment diluting supernatant antigen into serumand then quantitating the protein in solution indicates that serum haslittle or no effect on the assay. As can be seen in FIG. 2, like theresults shown in FIG. 1, serum samples from cancer patients reflect ahigher rate of cell death as indicated by the quantifiably higher levelsof antigen detected in these samples compared with those detected in thenormal blood serum samples.

Apoptosis and Necrosis

1. In this example, both apoptosis and necrosis were induced and theeffect of this injury on nuclear matrix protein release tested. In thisexperiment ME-180 cells (cervical tissue culture cell line) weresuspended into serum free medium. Necrosis was induced in one aliquot byfreezing the cells at -20° C., overnight. Cells were allowed to necrosefor seven days at 37° C. with 5% CO₂. Apoptosis was induced in a secondaliquot by serum deprivation for seven days at 37° C. with 5% CO₂.Supernatants from each aliquot then were harvested and assessed byimmunoradiometric assays using 302-22/302-18 ("22/18") and 204-41/107-7("41/7") antibody combinations. As shown in FIG. 3, the 22/18 assayshows specificity for apoptosis: ten times more 22/18 antigen isreleased by apoptotic cells (serum deprived, non-frozen) than bynecrotic cells (frozen). By contrast, the 41/7 assay measures an antigenthat is released substantially equally by both necrotic and apoptoticcells.

2. In this example apoptosis was induced by exposing MCF 7 cells (breastcarcinoma cell line, ATCC, Rockville, Md.) to Tumor Necrosis Factor (TNFalpha). MCF 7 cells were seeded into 12 individual T-25 flasks in 35 mlof Dulbecco Modified Media (DME) with 5% fetal calf serum and SeraXtend.The cells were grown at 37° C. and 5% CO₂ until 60-70% confluency. Cellsupernatants were removed and split into two groups of six:

Group 1: Control--cell supernatants were replaced with fresh media.

Group 2: TNF--Cell supernatants were replaced with fresh mediacontaining 500 U/ml recombinant human TNF-alpha (Cellular Products, Inc.(Buffalo, N.Y.).

At discrete intervals: 2 hours, 8 hours, 24 hours, 48 hours, and 72hours, supernatants were removed, spun down at 1200 rpm and frozen at-80° C. Cells remaining adhered were used for slides and PAP-stained(using standard cell staining techniques). Supernatants were assessed byimmunoradiometric assay using the 22/18 or 302.29/107.07 ("29/7")antibody combination. As seen in FIG. 4 cell treatment with TNF inducesthe release of nuclear matrix protein into the supernatant soon afterexposure (by 20 hours), whereas the untreated cells do not. FIG. 4A isassayed with the 22/18 combination; FIG. 4B with the 29/7 combination.In addition, histological evaluation of the stained cells verified thatthe TNF-treated cells showed the characteristics of apoptosis (chromatincondensation, cytoplasmic blebbing).

Dose vs. Density Experiment

Here, MCF7 cells (breast carcinoma, adherent) were grown in a one stagecell factory (Nunc) in DME complete (5% FCS+SerXtend) at 37° C. and 5%CO₂. These cells then were harvested, resuspended in serum free MinimalEssential Medium contain fungizone and gentamycin (MEM), counted, andseeded into T150 flasks with 35 mls serum free media at different celldensities.

The cells were allowed to die for 7 days at 37° C. and 5% CO₂. Thesupernatants were drawn off, spun down at 1000 rpm to remove cellfragments. All supernatants were assayed by immunoradiometric assaysusing the 22/18, and by the 41/7 antibody combination in an ELISA.

For all nuclear matrix proteins tested, release was essentially directlyrelated to cell density (See FIG. 5). The result is significant as thesepreliminary data suggest that nuclear matrix protein release isconsistent and quantifiable, and values detected can be correlated withcell number.

Cytotoxic Reagent Assay

1. In this experiment, the effect of increasing concentrations of TNF onthe release of the 41/7 nuclear matrix protein was investigated. MCF7tissue culture cells growing at 37° C. and 5% CO₂ in tissue culturemedium were exposed to different concentrations of TNF by replacing thetissue culture media with media containing TNF. As shown in FIG. 6, thequantity and rate of release of the 41/7 nuclear matrix protein isrelated to the dose of TNF added, particularly at doses greater than 1.0units/ml TNF (FIG. 6).

2. In this experiment, the effect of a known cancer chemotherapeuticagent (Doxorubicin, Sigma Inc.) on soluble nuclear matrix proteinrelease into extracellular fluid was assessed.

A standard curve for live cells was established with MCF7 cells (breastcarcinoma, adherent). Cells were seeded into 6 well plates in 2 mls ofDME complete (5% FCS+SerXtend) with 0.5 micrograms/ml mitomycin C(Sigma, Inc., Cincinnati) to prevent any cell growth.

After incubating overnight at 37° C., 200 μl of 3- 4, 5Dimethylthiazol-2-4! 2, 5-dipheny(tetrazoluim bromide), MTT, stocksolution (5 mg/ml MTT in phosphate-buffered saline, Sigma, Inc.) wereadded to each well. After the plates were incubated for four hours at37° C. and 5% CO₂ the supernatants were aspirated off and 4 mls of DMSO(dimethyl sulfoxide) were added to solubilize remaining crystals. Thiswas drawn off and the absorbance was read in the spectrophotometer at562 nm against a blank of straight DMSO.

Cells were treated with Doxorubicin as follows: MCF7 cells were seededinto 6 well plates at a density of 1.3×10⁵ cells/well in 2 mls DMEcomplete. After 72 hours of growth at 37° C. and 5% CO₂, thesupernatants were aspirated off and replaced with DME completecontaining 450 nM Doxorubicin.

At 1, 24, 48, 72, and 96 hours, the supernatants were removed and frozenat -20° C. to assay later. After replacing the supernatant with a fresh2 ml of DME complete, the number of live cells was determined using thesame procedure as for the standard curve. The cell supernatants wereassayed by ELISA, using the 41/7 antibody combination. The dose valuesobtained from these ELISAs were plotted along with standard curve datato observe correlations between the assay measurement of remaining livecells and nuclear matrix protein release (See FIG. 7)

As evidenced by FIG. 7, cell viability appears to be inversely relatedto nuclear matrix protein released.

TNF-Induced Release of Nuclear Matrix Protein

The present invention also may be used to prepare a NM preparation formcells by inducing their release from cells in soluble from with anappropriate agent such as TNF. Generally, a protocol similar to that ofthe cytotoxic reagent assay may be used to induce protein release. Cellsare grown essentially to confluency, and the supernatant then isreplaced with DME complete, 250 U/ml TNF. After an appropriateincubation period, the supernatant is drawn off and the soluble nuclearmatrix protein extracted from solution by standard protein separationmethods, for example, by chromatography on a cationic exchange column oran affinity column, such as an immunoaffinity column. Once purified, theNM preparation may be maintained as a frozen solution until ready to beused. The NM proteins so prepared can be used in immunoassay standardsand for the production of further monoclonal and polyclonal antibodies.It is also proposed that these proteins can be used to evaluate theirability to bind to other compounds and thus identify compounds capableof interfering with the nuclear function of particular nuclear matrixproteins. Compounds capable of specifically binding to and interferingwith the nuclear function of cancer-specific nuclear matrix proteins,for example, may be useful as cancer chemotherapeutic agents.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicted by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

What is claimed is:
 1. A method for evaluating the efficacy of a therapyassociated with cell death in a tissue, said method comprising the stepsof:(a) providing first and second fluid samples drawn from a patientafter initiation of a said therapy, wherein said first fluid sample isdrawn from said patient before said second fluid sample, and wherein atleast one of said samples contains soluble debris released from cells ofsaid tissue; (b) contacting said first and second fluid samples,separately, with a binding partner that binds specifically to a bodyfluid-soluble interior nuclear matrix protein having a specific bindingsite for said binding partner; (c) measuring, in each of said first andsecond fluid samples, a quantity per unit volume of said bodyfluid-soluble interior nuclear matrix protein recognized specifically bysaid binding partner, said quantity being indicative of the magnitude ofcell death in said tissue; and (d) comparing said quantities per unitvolume of said body fluid-soluble interior nuclear matrix protein insaid first and second fluid samples, wherein an increase in saidquantity of body fluid-soluble interior nuclear matrix protein in saidsecond fluid sample relative to said first fluid sample is indicative ofan increase in cell death and a decrease in said quantity of bodyfluid-soluble interior nuclear matrix protein in said second fluidsample relative to said first fluid sample is indicative of a decreasein cell death.
 2. The method of claim 1 wherein said therapy inducescell death.
 3. The method of claim 1, comprising the additional step of,prior to step (a), administering a therapeutic agent or procedure tosaid patient.
 4. The method of claim 3 wherein said therapeutic agent orprocedure is a cancer therapeutic agent or procedure.
 5. The method ofclaim 1, wherein said therapy comprises a treatment for a malignancy. 6.The method of claim 5, wherein said malignancy is selected from thegroup of malignancies consisting of a carcinoma, sarcoma, lymphoma and amyeloma.
 7. The method of claim 1 wherein said first and second fluidsamples provided in step (a) are selected from the group of samplesconsisting of blood, serum, plasma, urine, semen, spinal fluid, asciticfluid, peritoneal fluid, saliva, and sputum.
 8. The method of claim 1wherein said first and second fluid samples are samples of breastexudate.
 9. The method of claim 1 wherein said binding partner is anantibody.
 10. The method of claim 9 wherein said antibody is amonoclonal antibody.
 11. The method of claim 9 wherein said antibody isa polyclonal antibody.
 12. The method of claim 1, wherein said first andsecond fluid samples are urine samples.
 13. The method of claim 1,wherein said first and second fluid samples are serum samples.
 14. Amethod for evaluating the efficacy of a therapy associated with celldeath in a tissue, said method comprising the steps of:(a) providingfirst and second fluid samples from a patient, wherein said first fluidsample is drawn from said patient before administration of said therapyand said second fluid sample is drawn from said patient afteradministration of said therapy, and wherein at least one of said samplescontains soluble debris released from cells of said tissue; (b)contacting said first and second fluid samples, separately, with abinding partner that binds specifically to a body fluid-soluble interiornuclear matrix protein having a specific binding site for said bindingpartner; (c) measuring, in each of said first and second fluid samples,a quantity per unit volume of said body fluid-soluble interior nuclearmatrix protein recognized specifically by said binding partner, saidquantity being indicative of the magnitude of cell death in said tissue;and (d) comparing said quantities per unit volume of said bodyfluid-soluble interior nuclear matrix protein in said first and secondfluid samples, wherein an increase in said quantity of bodyfluid-soluble interior nuclear matrix protein in said second fluidsample relative to said first fluid sample is indicative of an increasein cell death and a decrease in said quantity of body fluid-solubleinterior nuclear matrix protein in said second fluid sample relative tosaid first fluid sample is indicative of a decrease in cell death. 15.The method of claim 14, comprising the additional step of administeringa therapeutic agent or procedure to said patient.
 16. The method ofclaim 15, wherein said therapeutic agent or procedure is a cancertherapeutic agent or procedure.
 17. The method of claim 14, wherein saidtherapy comprises a treatment for a malignancy.
 18. The method of claim17, wherein said malignancy is selected from the group of malignanciesconsisting of a carcinoma, sarcoma, lymphoma and a myeloma.
 19. Themethod of claim 14, wherein said first and second fluid samples providedin step (a) are selected from the group of samples consisting of blood,serum, plasma, urine, semen, spinal fluid, ascitic fluid, peritonealfluid, saliva, and sputum.
 20. The method of claim 14, wherein saidfirst and second fluid samples are samples of breast exudate.
 21. Themethod of claim 14, wherein said binding partner is an antibody.
 22. Themethod of claim 21, wherein said antibody is a monoclonal antibody. 23.The method of claim 21, wherein said antibody is a polyclonal antibody.24. The method of claim 14, wherein said first and second fluid samplesare urine samples.
 25. The method of claim 14, wherein said first andsecond fluid samples are serum samples.